Food supplement

ABSTRACT

A food supplement is composed of a water-insoluble extract of a microalga and at least one oil selected from medium-chain triglycerides (MCT), wherein the extract of microalga comprises at least 25 mg/g of one or several omega-3 type fatty acid(s), at least 5 mg/g of one or several xanthophyll(s), at least 0.5 mg/g of one or several sterol(s) and at least 1 μg/g of one or several phycoprostane(s). A method for preventing the apparition of age-related cognitive disorders, defined as a non-pathological decrease of the cognitive functions, or cognitive disorders in children or young adults having been subjected to a prenatal stress inducing non-pathological disorders, such as hyperactivity, attention and memory deficit, language retardation and anxious behavior, involves administration of an effective amount of the food supplement to a human in need thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-in-Part application of U.S.Non-provisional application Ser. No. 16/798,609, filed Feb. 24, 2020,which claims the benefit of FR Application No. 19/01820, filed Feb. 22,2019, both of which are incorporated by reference in their entiretyherein.

TECHNICAL FIELD

The present disclosure concerns a composition, as well as a foodsupplement based on fatty acids and xanthophylls and the applicationsthereof in particular to prevent the apparition of cognitive disordersin humans or animals.

BACKGROUND

The cognitive processes are defined as all cerebral functions allowingacquiring, processing, memorizing and using data originating from theenvironment in order to maximize the advantages and minimize thedrawbacks of the external constraints. Thus, the cognitive processes areimplemented during phases involving reasoning (giving rise to planning,organization, judgment), perception, recognition, language, emotions,memory and learning.

Light cognitive disorders, or cognitive fragilities, are defined asalterations of the cognitive functions without dementia. From a clinicalperspective, these disorders are associated to a 0.5 score in thecontext of an assessment via the CDR (Cognitive Drug ResearchComputerized Assessment System) test.

Amongst these cognitive disorders, a cognitive decline associated withageing and cognitive alterations induced by a prenatal stress are twophenomena that might intervene throughout the life of an individual.

The age-related cognitive decline is defined as a non-pathologicaldecrease in the cognitive functions such as the speed of informationprocessing, the attentional ability and especially the so-called working(or short-term) memory. These processes result from normal physiologicalmodifications directly related to the age. The age of the start of thisdecline is still controversial, but given the acceleration of the ageingof the world population, more than 20% of the world population beingmore than 60 years, and this percentage will be more than 30% in 2050,the age-related cognitive decline is amongst the major issues of theseupcoming decades, at global level, and specifically in developedcountries, in which it will considerably impact the economy (lessautonomy for elderly persons) and public policies.

At the opposite of the age composition pyramid, cognitive disorders mayaffect infants and young children following a prenatal stress. Indeed,for several years now, the influence of stress during some period of thepregnancy on the cognitive development of the unborn individual has beenstudied both in humans and in animals. Thus, in animals, mainly in rats,it has been demonstrated that a prenatal stress in the mother wouldinduce a progeny having an altered long-term memory.

It turns out that intense negative stimuli, stress, may inducenon-pathological alterations or decreases in the cognitive functions ofyoung children, manifested in an hyperactivity, an attention and memorydeficit, language retardation, more difficult temperaments and moregenerally behavioral alterations such as an anxious behavior, revealinga neurodevelopmental retardation and a decrease in the cognitiveabilities.

One amongst the presumed mechanisms of the expression of the prenatalstress into cognitive disorders, is based on the contact of the fetuswith large doses of so-called stress hormones, belonging to the familyof corticosteroids, such as cortisol. Yet, cortisol crosses theplacental barrier and, starting from a determined concentration, theprotective mechanisms of the fetus against the corticoids secreted bythe mother, are saturated, thereby bringing the fetus in contact withtoo considerable doses of cortisol which seem to have a negative effecton the cognitive development. Other complementary assumptions explainthe relationship between the prenatal stress and the cognitive disordersof the child, have been developed.

The stress concept may be defined according to different perspectivesuch as the biological approach: stress is then a series of metabolicreactions, following one or several exogenous factor(s), inducingphysiological or psychological changes (fear, anguish) in the organism.However, the stress concept and its impacts is largelyindividual-specific, and the response of the individual to a stress isalso defined from a psychological perspective. Henceforth, bearing inmind that an event is stressful only retrospectively, because of areaction that is specific to each individual, or because it may beobjectively stressful, it is difficult to act on the sources of prenatalstress. Added to this is the fact that pregnancy induces hormonal andpsychological changes increasing the sensitivity of the prospectivemother to every event likely to impact the well-being of the unborninfant.

The strictly therapeutic approach of stress or anxiety, in the form ofdrugs prescription, is dangerous in the case of a pregnant woman:numerous psychotropic drugs for the treatment of psychological disordersor anxiety have teratogenic effects with direct adverse effects on thefetus. This requires an assessment on a case-by-case basis, and thisapproach is used only in the case of clinical psychological disorders inthe pregnant woman and not in the case of so-called subjective stresses.

Thus, there is a major problem relating to the discovery of solutionsagainst the consequences of prenatal stress on cognitive disorders inchildren or young adults.

Different studies have shown the interest of a nutritionalsupplementation of so-called essential fatty acids, but also ofcarotenes and in particular xanthophylls, and even the combination ofsaid fatty acids and said carotenes, to prevent or at least limit thedecline of the cognitive functions. Food supplements or drugs have beendeveloped leading to positive results.

Thus, there is known according to the document WO2013/032333A1, acomposition based on omega-3 type fatty acids, in particular theeicosapentaenoic acid (EPA) and the docosahexaenoic acid (DHA),asthaxanthin and glycerophospholipids, which is recommended for theprevention or the treatment of different disorders and in particularcognitive disorders. Said ingredients are present in this composition inthe form of extracts of microalgae; in a preferred variant ofpreparation, they are obtained by formulation of two extractsoriginating from two different algae. The natural origin of theconstituents of the composition is a tremendous benefit. Nonetheless,the need for more effective compositions still exists, in particularwith regards to the aforementioned issues. Furthermore, it is importantto provide methods for preparing such compositions that are simple andreplicable.

BRIEF SUMMARY

The disclosure brings in a solution with a composition comprising one orseveral omega-3 type fatty acid(s) and one or several xanthophyll(s), aswell as one or several compound(s) of the family of sterols and one orseveral phycoprostane(s). It turned out that the combination of at leastone sterol and at least one phycoprostane with at least one omega-3 typefatty acid and at least one xanthophyll significantly increases theeffectiveness of a composition in the prevention of the apparition ofage-related cognitive disorders but also against those associated to aprenatal stress.

A composition of the disclosure comprises:

-   -   at least 25 mg/g, in particular at least 50 mg/g, of one or        several omega-3 type fatty acid(s),    -   at least 5 mg/g, in particular at least 10 mg/g, of one or        several xanthophyll(s),    -   at least 0.5 mg/g, in particular at least 1 mg/g, of one or        several sterol(s), and    -   at least 1 μg/g, in particular at least 2 μg/g, of one or        several phycoprostane(s).

In a primary indication, a composition of the disclosure may be used asa food supplement. Also, the disclosure concerns a food supplement whichcomprises at least 25 mg/g, in particular at least 50 mg/g, of one orseveral omega-3 type fatty acid(s), at least 5 mg/g, in particular atleast 10 mg/g, of one or several xanthophyll(s), at least 0.5 mg/g, inparticular at least 1 mg/g, of one or several sterol(s), and at least 1μg/g, in particular at least 2 μg/g of one or several phycoprostane(s).

The disclosure has an essential advantage in that all of theconstituents or ingredients hereinabove may be obtained from a naturalsource and in particular they may be extracted from one or severalmicroalga(e), and preferably from one single microalga. Of course, theconstituent(s) or ingredient(s) of a composition or of a food supplementof the disclosure may have a non-natural origin and be provided in theform of chemically synthesized products.

BRIEF DESCRIPTION OF THE DRAWINGS

The different embodiments of the disclosure are illustrated hereinbelowand their advantages set out in the following examples, with referenceto the following figures:

FIG. 1 is a representation of the effects of the supplement of thedisclosure on the locomotor activity, with the left-side diagramillustrating the effects on the spontaneous alternation deficits and theright-side diagram illustrating the effects on the locomotor activity.

FIG. 2 is a representation of the effects on the learning deficitsinduced by the D-Gal according to the MWM test.

FIG. 3 is a representation of the effects of the supplement and of DHAon the learning deficits induced by the D-Galactose.

FIG. 4 is a representation of the effects on the passive avoidancedeficits induced by the D-Galactose in mice, with the effects on thestep-down latency illustrated on the left-side diagram and on the escapelatency illustrated on the right-side diagram, measured during theretention period.

FIG. 5 is a representation of the effects of the supplement and of DHAon the lipid peroxidation induced by the D-Galactose.

FIG. 6 is a representation of the effects of the supplement and of DHAon the expression of TNF-α in the cortex and the plasma induced by theD-Galactose, with the effect on the cortex on the left-side diagram andthe effect on the plasma on the right-side diagram.

FIG. 7 is a representation of the effects of the supplement and of DHAon the expression of IL-6 in the cortex (left-side diagram) and theplasma (right-side diagram) induced by the D-Galactose.

FIG. 8 is a representation of the effect of the supplement on anxiety,in the test of locomotion at the center of the test space, day PPD46.

FIG. 9 is a representation of the effect of the supplement on therecognition memory, in the test of recognition of an object, day PPD47.

FIG. 10 is a representation of the effect of the supplement on therecognition memory, in the test of recognition of a new object.

FIG. 11 is a representation of the effects of the supplement of thedisclosure on the locomotor activity, with the left-side diagramillustrating the effects on the spontaneous alternation deficits and theright-side diagram illustrating the effects on the locomotor activity.

FIG. 12 is a representation of the effects on the learning deficitsinduced by the D-Gal according to the MWM test.

FIG. 13 is a representation of the effects of the supplement on thelearning deficits induced by the D-Galactose.

FIG. 14 is a representation of the effects on the passive avoidancedeficits induced by the D-Galactose in mice, with the effects on thestep-down latency illustrated on the left-side diagram and on the escapelatency illustrated on the right-side diagram, measured during theretention period.

FIG. 15 is a representation of the effects of the supplement on thelipid peroxidation induced by the D-Galactose.

FIG. 16 is a representation of the effects of the supplement on theexpression of TNF-α in the cortex and the plasma induced by theD-Galactose, with the effect on the cortex on the left-side diagram andthe effect on the plasma on the right-side diagram.

FIG. 17 is a representation of the effects of the supplement on theexpression of IL-6 in the cortex (left-side diagram) and the plasma(right-side diagram) induced by the D-Galactose.

DETAILED DESCRIPTION

Before disclosing the disclosure in more details, some terms used in thepresent text are defined.

The term «comprises» in the expression «a composition comprises» or «afood supplement comprises» means that the composition or the supplementmay incorporate any additional constituent or more, which is notspecifically mentioned, in any form and from any origin whatsoever. Italso covers a composition or a supplement that would contain only thelisted constituents and consequently the composition or the supplementwould consist of said constituents.

A food supplement is defined as one or several foodstuff(s) whosepurpose is to complete the normal diet of a human or of an animal, andwhich constitute a concentrated source of nutrients or other substanceshaving a nutritional or physiological effect alone or in combination; itis generally available in the form of doses, namely the packaging formssuch as gel capsules, pastilles, tablets, pills and other similar forms,as well as powder packs, ampoules of liquid, vials fitted with a dropperand the other similar forms of liquid or powder preparations intended tobe taken in units measured in a small amount.

Omega-3 type fatty acid(s) are a family of unsaturated fatty acids whosehydrocarbon chain has 4 to 36 atoms of carbon, in general from 14 to 36atoms of carbon, and whose double bond or whose first double bond,counted starting from the terminal methyl group of the chain, is on thethird carbon-carbon bond. The unsaturation(s) may be of the cis or transtype, independently from one another. The most representative acids arethe alpha-linolenic acid (ALA), the eicosapentaenoic acid (EPA) and thedocosahexaenoic acid (DHA), but the designation «omega-3 type fattyacids» is not restricted thereto. Furthermore, and in particular whenthe fatty acid(s) are from a natural origin, they may be extracted fromalgae and be in the form of free molecules but also in a derivative formsuch as an esterified form, for example in a mono-, di- ortri-esterified form, or in mixtures of these forms.

By xanthophylls, are defined the molecules belonging to the carotenoidsincluding one or several atom(s) of oxygen such as astaxanthin,canthaxanthin, vaucheriaxanthin, lutein, zeaxanthin, diadinoxanthin,neoxanthin, loroxanthin, siphonoxanthin, diatoxanthin, violaxanthin,dinoxanthin, flavoxanthin, α-cryptoxanthin, 6-cryptoxanthin andfucoxanthin. In particular, when the xanthophyll(s) are from a naturalorigin, they may be extracted from algae and be in the form of freemolecules but also in a derivative form such as an esterified form ofmono- or multi-esters, or in mixtures of these forms.

Sterol(s) are a family of well-known lipids having a sterane core whose3^(rd) position carbon carries a hydroxyl group, the latter may bemodified for example by an acetyl group. These include natural sterolsor phytosterols, and are grouped in the present text under the termphycosterols. Without limitation, as examples of phytosterols, mentionmay be made to 24-methylene cholesterol, 6-sitosterol, fucosterol,isofucosterol, saringosterol, oxocholesterol acetate, crinosterol, andmore particularly brassicasterol, stigmasterol and campesterol.

By phycoprostanes, it should be understood a family of lipids that arestructurally of the prostaglandin type, from a natural origin, resultingfrom non-directly enzymatic oxidations of the fatty acids naturallypresent within the microalgal biomasses. In particular, these compoundsare selected from phytoprostanes, isoprostanes and neuroprostanes,depending on the fatty acid that has undergone the oxidation(s). Thus,these compounds may originate from fatty acids such as the α-linolenicacid (ALA), the arachidonic acid (ARA), the eicosapentaenoic acid (EPA),or the docosahexaenoic acid (DHA). The phytoprostanes are mainly derivedfrom ALA and may be selected from 9-epi-9F1t-PhytoP, ent-16-epi-16-F1t-PhytoP, 9-F1t-PhytoP, ent-16B1t-PhytoP, ent-9L1t-PhytoP,16(RS)-16-A1t-PhytoP. The isoprostanes are mainly derived from ARA andEPA and may be selected from 15-E2t-IsoP, 15-F2t-IsoP,15-epi-15-F2t-IsoP, 5-F2t-IsoP, 8(RS)-8-F3t-IsoP. The neuroprostanes aremainly derived from DHA and may be selected from 4-F3t-NeuroP,10-F4t-NeuroP, 10-epi-10-F4t-NeuroP, 4(RS)-4-F4t-NeuroP,14(RS)-14-F4t-NeuroP, 20(R)-20-F4t-NeuroP.

By Medium-Chain Triglycerides (MCT), it should be understood esters ofglycerol and saturated fatty acids, whose hydrocarbon chain has 6 to 12atoms of carbon. They are naturally present in coconut palm oil such ascoconut oil, palm kernel oil and palm oil, but they may be obtained fromother greases or oils.

The present disclosure is described hereinafter in more details and itsvariants are disclosed.

Advantageously, a composition or a food supplement of the inventionpresents the following features, considered separately or in anycombination.

It comprises 25 to 250 mg/g, in particular 50 to 250 mg/g, of one orseveral omega-3 type fatty acid(s), 5 to 50 mg/g, in particular 10 to 50mg/g, of one or several xanthophyll(s), 0.5 to 20 mg/g, in particular 1to 20 mg/g, of one or several sterol(s) and 1 to 100 μg/g, in particular2 to 100 μg/g, of one or several phycoprostane(s).

It comprises 25 to 200 mg/g, in particular 50 to 200 mg/g, of one orseveral omega-3 type fatty acid(s), 5 to 30 mg/g, in particular 10 to 30mg/g, of one or several xanthophyll(s), 0.5 to 8 mg/g, in particular 1to 8 mg/g, of one or several sterol(s) and 1 to 50 μg/g, in particular 2to 50 μg/g, of one or several phycoprostane(s).

It comprises 25 to 170 mg/g, in particular 50 to 170 mg/g, of one orseveral omega-3 type fatty acid(s), 5 to 25 mg/g, in particular 10 to 25mg/g, of one or several xanthophyll(s), 0.5 to 6 mg/g, in particular 1to 6 mg/g, of one or several sterol(s) and 1 to 40 μg/g, in particular 2to 40 μg/g, of one or several phycoprostane(s).

Advantageously, a composition or a food supplement of the disclosurefurther contains at least one oil as a vehicle or support to facilitatethe expression of the active ingredients. Surprisingly, it has beenobserved that the production of a composition or of a food supplement isfacilitated when this oil is selected from medium-chain triglycerides(MCT). In particular, when the active ingredients are obtained from thesame microalga extract, an optimum homogenization is observed in suchoil. According to one variant, the medium-chain triglycerides (MCT) arefrom a natural origin and are brought by oil selected from coconut palmoil, palm kernel oil and palm oil; they may also be obtained or derivedfrom such oil.

Hereinafter, preferred formulations of a composition or of a foodsupplement of the disclosure are presented, these implementations may ofcourse be combined: the or at least one of the omega-3 type fatty acidsis selected from stearidonic acid (SDA), eicosapentaenoic acid (EPA),docosahexaenoic acid (DHA) and mixtures thereof;

-   -   the or at least one of the xanthophylls is fucoxanthin;    -   the or at least one of the sterols is selected from        phytosterols;    -   the or at least one of the phycoprostanes is selected from        phytoprostanes, isoprostanes and neuroprostanes.

A composition or a food supplement of the disclosure may comprise anyadditive allowing improving in particular the preservation, theappearance, the taste, the formulation thereof. Thus, one or severaladditive(s) as those selected from preservative agents, colorants,flavors, disintegration agents, lubricant agents, coating orencapsulation agents may be incorporated therein.

A major application of a composition of the disclosure is nutraceutical,thus, such a composition or food supplement as defined hereinabove isadvantageously in the form of gel capsules, capsules, tablets, pastillesor loose powder. Preferably, it is packaged in doses having a unitweight comprised between 1 mg and 1 g. In general, the galenic of thecomposition or of the supplement will be adapted to the consideredindividual and in particular depending on whether it is intended to achild or an adult.

A composition or a food supplement of the disclosure may be used toprevent the apparition of non-pathological age-related cognitivedisorders or non-pathological cognitive disorders in children or youngadults having been subjected to a prenatal stress. The “age-relatedcognitive disorders” are notably but not exclusively chosen from adeficit in spatial working memory, a deficit in attention and vigilance,a deficit in executive function, a deficit in episodic memory, and/or adeficit in perceptual and cognitive skills. The food supplementaccording to the disclosure has demonstrated its efficacy onto saiddeficits through human clinical trial(s), such as detailed in Example 5of the present application.

In particular, the food supplement may be used towards healthy olderhumans aged between 55 and 75 years old. More particularly, thedisclosure concerns a method for preventing the apparition ofage-related cognitive disorders, defined as a non-pathological decreaseof the cognitive functions, in order to improve calmness and reduceperceived stress. In the prevention of such age-related cognitivedisorders, the daily intake may be comprised between 2 to 16 mg/kg, inparticular 2 to 5 mg/kg of body weight. Such food supplement may beorally administrated daily during a period of between and included onemonth to 6 months. In particular, when the daily intake is 16 mg/kg, thefood supplement may be orally administrated during 1 month or during 3months, in particular to a healthy older humans aged between 55 and 75years old.

In the prevention of cognitive disorders in children or young adultshaving been subjected to a prenatal stress, the daily intake may becomprised between 0.05 to 0.1 mg/kg of body weight. It has been noticedthat an effect is observed even for very small daily intakes, providedthat the duration of the treatment is proportionally extended.

The disclosure also concerns the use of a microalga to prepare a foodsupplement as previously defined. One or several preferred microalga(e)are selected from any one of the following taxons Pinguiophyceae,Chrysophyceae, Bacillariophyceae, Mamiellophyceae, Prymnesiophyceae,Haptophyceae, Coccolithophyceae, Isochrysidaceae and Phaeodactylaceae.Advantageously, the microalga is Tisochrysis lutea or Phaeodactylumtricornutum. Such microalgae will be selected because an appropriateextraction leads to an extract whose composition meets the definition ofa composition of the disclosure. As example, such an extract maycomprise the following fraction of fatty acids: the fatty acids,expressed in weight percentages with respect to the total extract, arein the form of free fatty acids between 4 and 55%, in the form ofmonoacyl glycerol between 0.5 and 10%, in the form of diacylglycerolbetween 0.4 and 15% and in the form of triacyl glycerol between 2 and80%, in particular between 2 and 55%. These fatty acids are in the rangeof 2.5 to 20% (w/w), in particular in the range of 5 to 20% (w/w), ofthe fatty acids from the omega-3 series and between 0.5 and 5%, of thefatty acids from the omega-6 series. More specifically, in particular,the fatty acids are ALA (α-linolenic acid) between 0.5 and 10%, SDA(stearidonic acid) between 0.5 and 10%, EPA (eicosapentaenoic acid)between 0.05 and 20%, DHA (docosahexaenoic acid) between 0.1 and 10%.

As previously indicated, one of the interests of a composition or of afood supplement lies in the method of preparation thereof andspecifically the natural origin of its constituents all of which may beobtained from one single microalga.

Depending on the used microalga, the formulation of the composition maybe obtained directly with the extract. If such is not the case, theextract will be diluted to obtain the required concentrations accordingto the disclosure. Nonetheless, the disclosure is not limited to thisimplementation, thus, it may be considered that only one portion of theconstituents is from a natural origin, the others being obtained througha chemical synthesis and/or that the constituents from a natural origindo not originate from the same source, for example, they are notproduced from the same alga.

The measurement and the adjustment of the concentrations of the activeingredients in an extract, and in the composition or the obtained foodsupplement, are carried out using analytic techniques that fall withinthe general knowledges of those skilled in the art.

A method for producing a composition or a food supplement from amicroalga culture is described hereinafter in more details.

According to a variant of the disclosure, said organisms are microalgae,such as those belonging to the taxons Pinguiophyceae, Chrysophyceae,Bacillariophyceae, Mamiellophyceae, Prymnesiophyceae, Haptophyceae,Coccolithophyceae, Isochrysidaceae, Phaeodactylaceae. Thesephotosynthesis-capable microorganisms may be strictly autotrophic,mixotrophic or transiently heterotrophic. These organisms may beharvested in a natural or preferably cultivated environment.

By extract, it is referred to a fraction of the biomass derived from thephotosynthesis-capable organisms obtained by a method allowingobtaining, whether directly or indirectly, a composition of thedisclosure. These extracts have a composition, expressed in weightpercentage with respect to the total extract, in proteins comprisedbetween 5 and 30%, in lipids between 20 and 80%, between 0.05 and 2% ofsterols, in particular between 0.1 and 2% of sterols, between 0.1 and20% of chlorophyll.

More specifically, the lipophilic portion composing the extract,expressed in weight percentage with respect to the total extract, isconstituted by saturated fatty acids between 15 and 45%, polyunsaturatedacids between 5 and 20%, xanthophylls between 0.5 and 20%, in particularbetween 1 and 20%, and phycoprostane between 0.0001 and 0.007%, inparticular between 0.0002 and 0.007%.

For the production of the extract according to the disclosure, the cellsadvantageously consist of microalgae cells of the species Tisochrysislutea of the Isochrysidaceae family, or microalgae cells of the speciesPhaedactylum tricornutum of the Phaeodactylaceae family, produced bycarbon autotrophy.

Production Method of the Microalgae

The microalgae are ideally cultivated in a controlled manner withinadapted systems such as race-ways, open ponds or preferably in closedsystems such as photobioreactors. The used photobioreactors may be ofany existing type such as horizontal tubular photobioreactors, verticalsuch as so-called «green wall panel» systems, planar or column typephotobioreactors. Preferably, the production of the biomass will beperformed within a closed cultivation system, by zero-impact autotrophyon arable lands.

The production of the biomass is performed according to batch,fed-batch, continuous, semi-continuous, turbidostat or chemostat typeculture management practices.

Obtainment of the Extracts of these Microalgae

The extracts derived from these microorganisms are preferably obtainedafter concentration of the biomass by elimination of all or part ofwater using chemical or physical processes such as centrifugation,filtration, flocculation, sedimentation, whether coupled or not, atdrying steps by freeze-drying, vacuum-drying, drum-drying, atomizationor any other process allowing lowering the water content of the biomass.Complementarily to these steps, cellular lysis processes may beimplemented such as the applications of pressures, electric flows, shearforces, the use of enzymes, or any other processes allowingdestructuring the tissues, organs, cells or organelles.

The interesting compounds of the biomass are extracted according to asolid-liquid extraction type process, which may use hypercritical fluidsor subcritical fluids, which may involve co-treatments carried out inparallel or sequentially such as microwaves, ultrasounds, pressures,enzymes. The used solvents, whether pure or in a mixture, may consist ofacetone, hexane, ethyl acetate, methyltetrahydrofuran, heptane,methanol, natural or branched oils, ethanol or any other solventallowing extracting all or part of the hydrophobic and amphiphiliccompounds.

The solvent or the mixture of solvents is separated from the residualbiomass after extraction by centrifugation, filtration type processesand may be concentrated afterwards, or the solvent eliminated, bytechniques such as vacuum evaporation or any other technique allowingfor the selective evaporation of the considered solvent. The extractthus obtained has a lipophilic nature while including amphiphilicmolecules.

Formulation as a Food Supplement

The formulation of the extract is performed with compatible matrices,enabling the dissolution thereof in order to obtain a homogeneoussolution with the desired concentration of extract such as for example,vegetable oils such that olive oil, colza oil, linseed oil, sunfloweroil, grape seed oil, palm oil and preferably MCT oils, and composed toabout 70 weight % of a mixture of caprylic acid and capric acid, andpreferably selected from coconut oil or palm oil, the whole beingsupplemented with molecules allowing increasing stability such assynthetic or natural antioxidants. The weight rates of incorporation ofthe matrices/additives in order to obtain the supplement may reach 95weight % with respect to the weight of the food supplement, they aregenerally comprised between 15 and 80%, preferably between and 45%.

The extract, but preferably the formulated composition or the obtainedsupplement, may be formulated in form of soft capsules, or may beformulated in form of powder, by any technique enabling themicro-encapsulation of the aqueous solution involving, or not, a supportor a matrix allowing, or not, a homogeneous dispersibility thereofwithin a drinkable polar solution.

The extract or the supplement may be used alone or as an ingredientwithin a food complementation.

This disclosure includes at least the following individual embodiments.These and other embodiments of the food supplement described herein arenot mutually exclusive, and are independently combinable.

In some embodiments, a food supplement comprises a water-insolubleextract of a microalga and at least one oil selected from medium-chaintriglycerides (MCT), wherein the extract of microalga comprises at least25 mg/g of one or several omega-3 type fatty acid(s), at least 5 mg/g ofone or several xanthophyll(s), at least mg/g of one or several sterol(s)and at least 1 μg/g of one or several phycoprostane(s).

The extract can comprise 25 to 250 mg/g of one or several omega-3 typefatty acid(s), 5 to 50 mg/g of one or several xanthophyll(s), 0.5 to 20mg/g of one or several sterol(s), 1 to 100 μg/g of one or severalphycoprostane(s).

The extract can also comprise 25 to 200 mg/g of one or several omega-3type fatty acid(s), 5 to 30 mg/g of one or several xanthophyll(s), 0.5to 8 mg/g of one or several sterol(s) and 1 to 50 μg/g of one or severalphycoprostane(s).

The extract can also comprise 25 to 170 mg/g of one or several omega-3type fatty acid(s), 5 to 25 mg/g of one or several xanthophyll(s), 0.5to 6 mg/g of one or several sterol(s) and 1 to 40 μg/g of one or severalphycoprostane(s).

The medium-chain triglycerides (MCT) can be selected from coconut palmoil and palm oil.

The one or at least one of the omega-3 type fatty acids can be selectedfrom ALA (α-linolenic acid), stearidonic acid (SDA), eicosapentaenoicacid (EPA), docosahexaenoic acid (DHA), and mixtures thereof.

The at least one of the xanthophylls can be fucoxanthin.

The or at least one of the sterols is selected from phytosterols.

The or at least one of the phycoprostanes can be selected fromphytoprostanes, isoprostanes and neuroprostanes.

The food supplement can further comprise at least one additive selectedfrom preservative agents, colorants, flavors, disintegration agents,lubricant agents, coating or encapsulation agents.

The food supplement can be in the form of gel capsules, capsules,tablets, pastilles or loose powder.

The food supplement can be packaged in doses having a unit weightcomprised between 10 mg and 1 g.

The microalga can be selected from any one of the taxons Pinguiophyceae,Chrysophyceae, Bacillariophyceae, Mamiellophyceae, Prymnesiophyceae,Haptophyceae, Coccolithophyceae, Isochrysidaceae and Phaeodactylaceae.

The microalga can also be Tisochrysis lutea or Phaeodactylumtricornutum.

In some embodiments, a method for preventing the apparition ofage-related cognitive disorders, defined as a non-pathological decreaseof the cognitive functions, or cognitive disorders in children or youngadults having been subjected to a prenatal stress inducingnon-pathological disorders, such as hyperactivity, attention and memorydeficit, language retardation and anxious behavior, comprises theadministration of an effective amount of the food supplement to a humanin need thereof.

The age-related cognitive disorders can be chosen from deficit inspatial working memory, deficit in attention and vigilance, deficit inexecutive function, deficit in episodic memory, and deficit inperceptual and cognitive skills.

The effective amount of the food supplement can improve calmness andreduce perceived stress.

The method can comprise the administration of an effective amount of thefood supplement to a healthy older human aged between 55 and 75 yearsold.

The effective amount can correspond to a daily intake of between 2 to 16mg of food supplement/kg of body weight.

The food supplement can be orally administrated during between 1 monthto 6 months.

The effective amount can correspond to a daily intake of between 0.05 to0.1 mg of food supplement/kg of body weight.

EXAMPLES Example 1: Formulation of an Extract Containing theConstituents of a Composition of the Disclosure

An extract is obtained according to any of the above-describedtechniques from the microalga Phaeodactylum tricornutum. In particularits process of preparation is as follows: the P. tricornutum microalgaeextract is obtained from the biomass of a microalgae culture of a strainoriginating in France. The biomass is first centrifuged and thenfiltered to remove the water. A solid-liquid extraction step is thenimplemented. The extract is thus obtained by extraction in awater/ethanol mixture in a proportion of 40/60 (w/w) to 1/100 (w/w), ata temperature of 20° C., i.e. at room temperature, for a period of 2hours. It is then filtered and then dried by freeze-drying. It comes inpowder form.

It is water-insoluble and is highly viscous preventing any handling atroom temperature.

The extract and the palm oil are kept at room temperature (25±1° C.) for24 h before preparation. The extract is transferred in a centrifuge tubeincluding the oil such that the final net mass of the mixture is about 5g and the mass proportion is such that the extract consists of 25% ofthe overall net mass of the mixture. The mixture is stirred for one unitusing a so-called vortex mixing device. Stirring is repeated three timesfor each mixture. A homogeneous mixture is obtained.

Example 2: Test of a Natural Extract of the Microalga Tisochrysis Luteain the Context of the In Vivo Model on the Attenuation of the DeficitsInduced by the Age-Related Cognitive Decline

The food supplement of the disclosure is prepared from a Tisochrysislutea extract which comprises in mg/g:

-   -   Omega-3 type fatty acids (ALA, SDA, EPA, DHA): 152.6±14.4;    -   Fucoxanthin: 20.0±4.0;    -   Sterols: 4.9±0.8;    -   Phycoprostane: 0.035±0.007.

The supplement is obtained by addition of coconut oil in a proportion of360 mg±10 mg/g to said extract.

The supplement is incorporated into kibbles according to 3 differentformulations such that the final concentrations of DHA in these batchesof kibbles are equal to 0.5, 1.5 and 3.0% (w:w).

A commercial oily extract of microalgae comprising, as a fat fraction,only the fatty acids DHA 77% (w:w) and EPA 3% (w:w) is also tested; itis also incorporated into a batch of kibbles such that the finalconcentration of DHA in this batch of kibbles is equal to 3.1% (w:w).

An additional batch of kibbles is formulated only with the coconut oil,such that the vehicle concentration is equivalent to that of the otherbatches, namely 0.01% (w:w).

The five batches of kibbles thus obtained are referenced as described inTable 1 hereinbelow.

TABLE 1 Formulated kibbles Reference [DHA] in % (w:w) Coconut oil A1 0Supplement A2 0.5 Supplement A3 1.5 Supplement A4 3.0 Commercial oilyextract A5 3.1

The considered in vivo model is the D-Galactose model applied to micewhich is suitable for the study of the age-related cognitive decline.Indeed, this model mimics numerous behavioral and molecularcharacteristics of the cerebral ageing in rodents' models.

The D-Galactose is administered subcutaneously in a daily proportion of150 mg/kg of mice wet weight, and the food supplement hereinabove isincorporated into a pellet, according to the following pattern:

-   -   Between day −14 and day 51, the supplement is administered by        incorporation into food pellets;    -   Between day 01 and day 51, the D-Galactose is administered        subcutaneously, five days a week;    -   Between days 43 and 51, three different behavioral tests are        used to monitor the effects of the test compounds.

The effectiveness of the supplement is assessed according to thefollowing parameters: improvement of the learning deficits (spatialworking memory: spontaneous alternation in the Y labyrinth according tothe Y-maze test; spatial memory by the so-called «Morris Water Maze» andlong-term contextual memory in the passive avoidance test), lipidperoxidation (LPO) rate in the hippocampus and effect on theneuro-inflammation markers IL6 and TNFα.

Improvement of the learning deficits

-   -   On day 43, all animals have been tested for the spontaneous        alternation performance in the Y-maze (YM) test, via a spatial        working memory index;    -   From day 44 to day 49, all animals have been tested for the        spatial memory in the Morris Water Maze (MWM) test, via a        spatial memory index;    -   From day 44 to day 49, all animals are tested via the MWM test        to assess the spatial working memory;    -   On days 50 and 51, the long-term contextual memory of the        animals is assessed using the step-by-step type passive        avoidance process (STPA), through exercise and retention        sessions, respectively;    -   On days 50 and 51, all animals are tested for the STPA task.

Lipid Peroxidation (LPO) Rate in the Hippocampus and Effect on theNeuro-Inflammation Markers IL6 and TNFα

On the 51th day, after the behavioral tests, the animals have beeneuthanized. For all animals, trunk blood is collected and centrifuged torecover plasma and the brain is rapidly collected. The hippocampus andthe cortex are dissected, the hippocampus is then used to determine thelipid peroxidation rates by a colorimetric method; the hemi-frontalcortex and the plasma are used to determine the level of theinflammatory biomarkers interleukin-6 (IL-6) and tumor necrosis factoralpha (TNF-α)

The quantification of the lipid peroxidation (LPO) rates has beencarried out according to the modified and adapted procedure ofHermes-Lima et al. This method measures the capacity of the peroxidizedlipids of the brain to oxidize a ferrous oxide and xylenol orangecomplex, set out in the presence of cumene hydroperoxide (HPC). Thelipid peroxidation level is determined in HPC-equivalent according tothe formula:

HPCE=A5801/A5802×[HPC(nmol)]

-   -   and expressed in HPC-equivalent per wet tissue weight and in        percentage with respect to the data obtained for the control        group (D-Galactose+vehicle).

The IL6 and TNFα contents are quantified by means of ELISA tests withthe following kits:

-   -   For the quantification of IL6: ThermoScientifique, EM2IL6    -   For the quantification of TNFα: ThermoScientifique, EMTNFA    -   For all tests, the cortex is homogenized after defrosting in a        buffer of 50 mM Tris-150 mM NaCl, pH 7.5, and sonicated for        20 s. After centrifugation (16 100 g for min, 4° C.), a        supernatant or plasma are used for the ELISA tests in compliance        with the instructions of the manufacturer of the ELISA tests.        For each test, the absorbance is read at 450 nm and the        concentration of the sample is calculated using the standard        curve. The results are expressed in pg of marker per mg of wet        tissue.

All values, except the passive avoidance latencies, are expressed as anaverage more or less the standard deviation of the measurement.Statistical analyses are performed separately for each compound using aunidirectional ANOVA (value F), followed by a Dunnett post-hoc multiplecomparison test. The passive avoidance latencies do not follow aGaussian distribution, since the upper limit times are fixed.

Hence, they are analyzed using a Kruskal-Wallis non-parametric ANOVA(value H), followed by Dunn multiple comparison test. The values withp<0.05 are considered as statistically significant.

The tests are performed on 60 male mice, distributed in 6 groups of 10mice, amongst which the group 1 is the negative control group and thegroups 2-6 are the positive control groups:

-   -   the group 1 is the group to which a subcutaneous saline solution        is administered instead of D-Galactose and kibbles A1;    -   the group 2 is the group to which D-Galactose and kibbles A1 are        administered;    -   the group 3 is the group to which D-Galactose and kibbles A2 are        administered;    -   the group 4 is the group to which D-Galactose and kibbles A3 are        administered; and    -   the group 5 is the group to which D-Galactose and kibbles A4 are        administered; and    -   the group 6 is the group to which D-Galactose and kibbles A5 are        administered.

The calculation of the human equivalent daily dose, from the daily dosetested in mice is defined as follows by the FDA (Guidance, 2005): thedaily dose in human expressed in mg/kg of body weight (HED Human) isequal to the daily dose in the animal expressed in mg/kg of body weight(HED Animal) multiplied by the ratio of the safety factor (Km Animal) inthe considered animal and of the safety factor for humans (Km Human). KmHuman is equal to 37 and Km Mice is equal to 3.

Effects on the Spatial Memory in the Y-Maze Spontaneous AlternationTest:

The results are represented in FIG. 1 , the first diagram (to the left)illustrating the effects of the supplement of the disclosure on thespontaneous alternation deficits and the second diagram (to the right)illustrating the effects of the supplement of the disclosure on thelocomotor activity.

In FIG. 1 : LOW, low dose of the supplement (A2); MED, medium dose ofthe supplement (A3); HI, high dose of the supplement (A4); N iscomprised between 9 and 10 depending on the groups; * p<0.05, ***p<0.0001 vs. the saline solution/group Veh, #p<0.05, ##p<0.01,###p<0.0001 vs. the group D-GAL 150/group Veh; Dunnett test.

It is observed that the treatment with D-Galactose has significantlyaltered the spatial working memory, in comparison with the mice treatedwith the saline solution. The supplement A2 has not demonstrated anyeffect on the alternation behavior. The supplement A3 has quitesignificantly but partially attenuated the deficits induced by thechronic intoxication with D-Galactose. The supplement A4 has quitesignificantly and completely attenuated the deficits induced by thechronic intoxication with D-Galactose.

The treatment with DHA alone (according to A5) has quite significantlybut partially alleviated the deficits induced by the chronicintoxication with D-Gal.

Surprisingly, it turns out that the preventive treatment with thesupplement according to the disclosure has a more considerable positiveeffect (quite significant and complete attenuation of the deficits) incomparison with the treatment with DHA alone (quite significant andpartial attenuation of the deficits), and that for the same dose of DHA.

Effects on the Learning Deficits Induced by the D-Gal According to theMWM Test:

The results are represented in FIG. 2 .

In FIG. 2 : LOW, low dose of the supplement (A2); MED, medium dose ofthe supplement (A3); HI, high dose of the supplement (A4); N iscomprised between 9 and 10 depending on the groups; * p<0.05, ** p<0.01,*** p<0.0001 vs. the saline solution/group Veh, ##p<0.01, ###p<0.0001vs. the group D-GAL 150/group Veh; Bonferroni multiple comparison testafter bidirectional ANOVA.

The chronic intoxication with D-Galactose has considerably altered thespatial learning, in comparison with the negative control group (salinesolution/vehicle).

The supplement A2 has not demonstrated any effect on the alternationbehavior.

The supplement A3 has quite significantly but partially attenuated thedeficits induced by the chronic intoxication with D-Galactose.

The supplement A4 has quite significantly and completely attenuated thedeficits induced by the chronic intoxication with D-Galactose.

The DHA alone according to A5 has quite significantly but partiallyalleviated the deficits induced by the chronic intoxication withD-Galactose.

Surprisingly, it turns out that the preventive treatment with thesupplement according to the disclosure at the dose A4 has a moreconsiderable positive effect (quite significant and complete attenuationof the deficits) in comparison with the treatment with DHA alone (quitesignificant and partial attenuation of the deficits), and that for thesame dose of DHA.

Effects of the Supplement and of the DHA on the Learning DeficitsInduced by the D-Galactose

The results are represented in FIG. 3 .

In FIG. 3 : LOW, low dose of the supplement (A2); MED, medium dose ofthe supplement (A3); HI, high dose of the supplement (A4); N iscomprised between 9 and 10 depending on the group; *** p<0.0001 vs. thesaline solution/group Veh,/Veh; ###p<0.0001 vs. the group D-GAL 150/Veh;Bonferroni multiple comparison test after bidirectional ANOVA.

The chronic intoxication with D-Galactose has considerably altered thespatial learning, in comparison with the negative control group (salinesolution/vehicle).

The supplement A2 has not demonstrated any effect on the alternationbehavior.

The supplement A3 has quite significantly but partially attenuated thedeficits induced by the chronic intoxication with D-Galactose.

The supplement A4 has quite significantly and completely alleviated thedeficits induced by the chronic intoxication with D-Galactose.

The treatment with DHA alone according to A5 has quite significantly butpartially alleviated the deficits induced by the chronic intoxicationwith D-Galactose.

Surprisingly, it turns out that the preventive treatment with thesupplement according to the disclosure at the dose A4 has a moreconsiderable positive effect (quite significant and complete attenuationof the deficits) in comparison with the treatment with DHA alone (quitesignificant and partial attenuation of the deficits), and that for thesame dose of DHA. In addition, the preventive treatment with thesupplement at the dose A3 has an effect (quite significant and partialattenuation of the deficits) that is identical to the treatment with DHAalone while the latter is two times more concentrated in DHA.

Effects on the Passive Avoidance Deficits Induced by the D-Galactose inMice

The results are represented in FIG. 4 , with the effects of thesupplement of the disclosure on the step-down latency illustrated on theleft-side diagram and on the escape latency illustrated on theright-side diagram, measured during the retention period.

In FIG. 4 : LOW, low dose of the supplement (A2); MED, medium dose ofthe supplement (A3); HI, high dose of the supplement (A4). N iscomprised between 9 and 10 depending on the groups; *** p<0.0001 vs. thesaline solution/group Veh, ###p<0.0001 vs. the group D-GAL 150/groupVeh; Dunnett test.

The chronic intoxication with D-Galactose has considerably altered thelong-term contextual working memory, in comparison with the negativecontrol group (saline solution/vehicle).

The supplement A2 has not demonstrated any effect on the long-termcontextual memory.

The supplement A3 has allowed attenuating the deficits induced by thechronic intoxication with D-Galactose in a non-significant manner.

The supplement A4 has quite significantly and completely attenuated thedeficits induced by the chronic intoxication with D-Galactose.

The treatment with DHA alone (according to A5) has allowed attenuatingthe deficits induced by the chronic intoxication with D-Galactose in anon-significant manner.

Surprisingly, it turns out that the preventive treatment with thesupplement of the disclosure at the dose A4 has a more considerablepositive effect (quite significant and complete attenuation of thedeficits) in comparison with the treatment with DHA alone(non-significant attenuation of the deficits), and that for the samedose of DHA. In addition, the preventive treatment with the supplementat the dose A3 has an effect (non-significant attenuation of thedeficits) that is identical to the treatment with DHA alone while thelatter is two times more concentrated in DHA.

Effects of the Supplement and of the DHA on the Lipid PeroxidationInduced by the D-Galactose

The results are represented in FIG. 5 .

In FIG. 5 : LOW, low dose of the supplement (A2); MED, medium dose ofthe supplement (A3); HI, high dose of the supplement (A4). N iscomprised between 9 and 10 depending on the groups; ** p<0.01, ***p<0.0001 vs. the saline solution/group Veh, ##p<0.01, ###p<0.0001 vs.the group D-GAL 150/group Veh; Dunnett test.

The chronic intoxication with D-Galactose has considerably increased theoxidative stress, in comparison with the negative control group (salinesolution/vehicle).

The supplement A2 has not demonstrated any effect on the lipidperoxidation induced by the chronic intoxication with D-Galactose.

The supplement A3 has quite significantly but partially reduced theoxidative stress induced by the chronic intoxication with D-Galactose.

The supplement A4 has quite significantly and completely reduced theoxidative stress induced by the chronic intoxication with D-Galactose.

The treatment with DHA alone according to A5 has not demonstrated anyeffect on the oxidative stress induced by the chronic intoxication withD-Galactose.

Surprisingly, it turns out that the preventive treatment with thesupplement of the disclosure at the dose A4 has a more considerablepositive effect (quite significant and complete attenuation of theoxidative stress) in comparison with the treatment with DHA alone, andthat for the same dose of DHA.

Effects of the Supplement and of the DHA on the Induced Expression ofTNF-α in the Cortex and the Plasma by the D-Galactose

The results are represented in FIG. 6 , with the effect on the cortex onthe left-side diagram and the effect on the plasma on the right-sidediagram.

In FIG. 6 : LOW, low dose of the supplement (A2); MED, medium dose ofthe supplement (A3); HI, high dose of the supplement (A4); N iscomprised between 9 and 10 depending on the groups; *** p<0.0001 vs. thesaline solution/group Veh, ###p<0.0001 vs. the group D-GAL 150/groupVeh; Dunnett test.

The chronic intoxication with D-Galactose has considerably increased ina significant manner the TNF-α in the cortex and the plasma, incomparison with the negative control group (saline solution/vehicle).

The supplements A2 and A3 have quite significantly but partially reducedthe increase of the TNF-α induced by the chronic intoxication withD-Galactose in the cortex and the plasma.

The supplement A4 has quite significantly and completely reduced thelevel of the TNF-α in the cortex and the plasma.

The treatment with DHA alone according to A5 has quite significantly butpartially reduced the increase of the TNF-α induced by the chronicintoxication with D-Galactose in the cortex and the plasma.

Surprisingly, it turns out that the preventive treatment with thesupplement HI (A4) has a more considerable positive effect (quitesignificant and complete attenuation of the increase of the TNF-α in thecortex and in the plasma) in comparison with the treatment with DHAalone, and that for the same dose of DHA. In addition, the preventivetreatments with the supplement at the doses A2 and A3 have effects inthe case of the cortex and the plasma (quite significant attenuations ofthe increases) that are identical to the treatment with DHA alone whilethe latter is respectively six and two times more concentrated in DHA,in comparison with the preventive treatments with the supplement at thedoses A2 and A3, respectively.

Effects of the Supplement and of the DHA on the Induced Expression ofIL-6 in the Cortex and the Plasma by the D-Galactose

The results are represented in FIG. 7 , with the effect on the cortex onthe left-side diagram and the effect on the plasma on the right-sidediagram.

In FIG. 7 : LOW, low dose of the supplement (A2); MED, medium dose ofthe supplement (A3); HI, high dose of the supplement (A4); N iscomprised between 9 and 10 depending on the groups; *** p<0.0001 vs. thesaline solution/group Veh, ###p<0.0001 vs. the group D-GAL 150/groupVeh; Dunnett test.

The chronic intoxication with D-Galactose has considerably increased ina significant manner the IL-6 in the cortex and the plasma, incomparison with the negative control group (saline solution/vehicle).

The low-dose supplement (A2) has not demonstrated any effect on theconcentration of IL-6 induced by intoxication with D-Galactose.

The medium-dose supplement (A3) has quite significantly but partiallyreduced the increase of the IL-6 content induced by the chronicintoxication with D-Galactose in the cortex and the plasma.

The high-dose supplement (A4) has quite significantly and completelyreduced the IL-6 content in the cortex and the plasma, induced by thechronic intoxication with D-Galactose in the cortex and the plasma.

The treatment with DHA alone according to A5 has quite significantly butpartially reduced the increase of the IL-6 induced by the chronicintoxication with D-Galactose in the cortex and the plasma.

Surprisingly, it turns out that the preventive treatment with thesupplement HI according to A4 has a more considerable positive effect(quite significant and complete attenuation of the increase of the IL-6in the cortex and in the plasma) in comparison with the treatment withDHA alone (quite significant and partial attenuation of the increase ofthe IL-6 in the cortex and in the plasma), and that for the same dose ofDHA. In addition, the preventive treatment with the supplement at thedose A3 has an effect in the case of the cortex (non-significantattenuation of the deficits) that is identical to the treatment with DHAalone while the latter is two times more concentrated in DHA.

In conclusion:

The chronic intoxication with D-Galactose has quite significantlyinduced an alteration of the spatial working memory, of the long-termcontextual memory and has prejudiced the spatial learning. Thebehavioral alterations are also related to biochemical alterationsmanifested in an increase of oxidative stress and in an induction ofneuro-inflammatory processes.

The preventive treatment with the supplement of the disclosure isdose-dependent and has quite significantly and completely attenuated inthe case of the strongest tested dose (the supplement D4) the deficitsinduced by the chronic intoxication with D-Galactose manifested in abehavioral alteration, an increase of oxidative stress and an activationof neuro-inflammatory processes.

The treatment with DHA alone (A5), and at an equivalent dose of DHA incomparison with the treatment with the supplement HI, has quitesignificantly but partially reduced the deficits induced by the chronicintoxication with D-Galactose manifested in a behavioral alteration, anincrease of oxidative stress and an activation of neuro-inflammatoryprocesses.

Surprisingly, the preventive treatment by the supplement issignificantly more effective at an equivalent dose of DHA than thepreventive treatment with DHA alone, with regards to the attenuation ofthe age-related cognitive decline on a murine model caused by a chronicintoxication with D-Galactose. In addition, the preventive treatment bythe supplement, for doses of DHA that are two times lower, has positiveeffects which are identical to the treatment with DHA alone, while thelatter is two times more concentrated in DHA, in the case of thedecrease of the oxidative stress measured in the cortex (IL-6 andTNF-α), in the plasma (TNF-α), in the case of the long-term contextualmemory and in the case of spatial learning. And, the preventivetreatment by the supplement has positive effects which are identical tothe treatment with DHA alone, while the latter is six times moreconcentrated in DHA, in the case of the decrease of the oxidative stressmeasured in the cortex (IL-6 and TNF-α) and the plasma (TNF-α).

Thus, by applying the formula for calculating the human equivalent dailydose, a preventive treatment of the age-related cognitive decline may bedefined with the daily intake of 2 to 5 mg of supplement/kg of bodyweight.

Example 3: Test of an Extract of the Microalga Tisochrysis Lutea inYoung Female Rats Suffering from a Prenatal Stress

In this example, the resolution of the cognitive deficits, the anxiousbehavior and the alteration of the recognition memory, induced in youngfemale rats after a prenatal stress of the ancestry thereof, via theadministration of a supplement based on an extract of the microalgaTisochrysis lutea corresponding to that used in Example 2, is studied

Equipment and Methods

The model used in this example is an acknowledged model for inducingprenatal stress in rats by immobilizing the pregnant female in acylinder under a harsh lighting.

Pregnant female rats have been randomly assigned to prenatal stressgroup (SP) or to control groups (NS), individually put in plastic-madebreeding cages, and have had ad libitum access to food and water, exceptduring the behavioral tests time. The conditions within the cages are asfollows: photoperiod 12 h of light/12 h of darkness cycle (light turnedon at 7 AM), in a room at constant temperature (21° C.) and constanthumidity (50%).

The prenatal stress procedure has been performed as described by Meunieret al. (2004). The immobilizations of the female rats have been theobject of a semi-random constraining procedure. The animals have beenplace and retained in Plexiglas-made transparent ferret retainers (20 cmlong, 7 cm diameter) under a bright light for a total period of 90minutes a day, for 4 consecutive days. For the stress to be asunpredictable as possible, the 90 minutes forced immobilization periodhas been administered in the following manner: one single 90 min phase,two min phases spaced by 4 h, two 60 and 30 min phases spaced by 4 h, orthree 30 min phases spaced by 4 and 1 h, and that, at different times ofthe day.

Control mothers have also been manipulated, but have never been placedin the ferret retainers.

The treated female rats have been allowed to clear themselves naturallyfrom the trap of the ferret retainers as of the day 1 after the birth(PPD1).

The litters have been weaned on PPD21. The rats have been separated fromthe mothers, identified according to their gender, weighted and rats ofthe same gender have been distributed in cages (3 rats per cage). Theyoung rats within the same cage originated from different litters, inorder to avoid any possible litter-related effect.

The conditions within the cages are as follows: photoperiod 12 h oflight/12 h of darkness cycle (light turned on at 7 AM), in a room atconstant temperature (21° C.) and constant humidity (50%), with adlibitum access to food and water, except during the behavioral teststime.

In each cage, the animals have received the same treatment. The animalshave been tested randomly and in a double-blind manner.

Forty-eight (48) female rats have been used and grouped into four groupsof animals, constituted in the following manner:

-   -   The group 1 is composed by 12 naïve female rats, that is to say        whose ancestry has not been subjected to a prenatal stress, and        receiving only 200 μL a day of the vehicle solution (reference:        NS/Vehicle). Hence, this group is the control group;    -   The group 2 is composed by 12 naïve female rats, that is to say        whose ancestry has not been subjected to a prenatal stress, and        receiving 200 μL a day of the supplement (reference:        NS/Supplement);    -   The group 3 is composed by 12 female rats whose ancestry has        been subjected to a prenatal stress, and receiving 200 μL a day        of the vehicle solution (reference: SP/Vehicle);    -   The group 4 is composed by 12 female rats whose ancestry has        been subjected to a prenatal stress, and receiving 200 μL a day        of the supplement (reference: SP/Supplement);    -   The effectiveness of the supplement has been assessed 6 weeks        after birth.

The supplement (one dose) has been administered by gavage once everyday, 5 days a week. The administrations have started after weaning,namely after the postpartum day (PPD) 25, and has lasted until PPD46.

The daily intake was 25.7 mg of supplement per kg of rat body weight.

The animals have been subjected to behavioral tests during the periodbetween the days PPD46 and PPD48, namely outside the period of treatmentwith the vehicle or the supplement. Hence, the effects that aretherefore observed during the behavioral tests will be due to atreatment that is preventive in nature.

The behavioral tests are divided into one anxiety assessment session andtwo objects recognition sessions. The sessions are defined as follows:

-   -   Session 1, PPD 46: The rats have been individually placed in a        square open space (50 cm×50 cm×50 cm×50 cm) made of blue-colored        Plexiglas with a floor equipped with infrared light-emitting        diodes. The rats have been accustomed to the test space during a        10 minute session and their displacements captured by an        infrared camera and analyzed using the Ethovision® (Noldus)        software. The activity has been analyzed according to the        overall covered distance (m), and according to the percentage of        presence in the 25 cm×25 cm central area defined by the        software/these data report on the intensity of the anxious        behavior (38).    -   Session 2, PPD 47: Two identical objects (50 mL plastic-made        Eppendorf tube) have been placed at determined locations (on two        opposite edges of the central area). Each rat has been placed        within the test space and the exploratory activity recorded        during a 10 minute session. The activity has been analyzed in        terms of number of contacts with the objects and of duration of        the contacts.    -   Session 3, PPD 48: The object of session 2 has been replaced        with a new object (a plastic-made bottle cap) whose shape,        texture, color differ from those of the familiar object. Each        rat has been replaced in the test space and the exploratory        activity has been recorded during a 10 minute session. The        activity has been the object of an analysis similar to that        described in session 2.

The preferential exploration index has been calculated as the ratio ofthe number (or duration) of contacts with the object of session 2, tothe overall number (or duration) of contacts with both objects.

All values are expressed as an average more or less the standarddeviation of the measurement. Statistical analyses are performedseparately for each compound using a unidirectional ANOVA (value F),followed by a Dunnett post-hoc multiple comparison test.

The calculation of the human equivalent daily dose, from the daily dosetested in rats is defined as follows by the FDA (Guidance, 2005): thedaily dose in human expressed in mg/kg of body weight (HED Human) isequal to the daily dose in the animal expressed in mg/kg of body weight(HED Animal) multiplied by the ratio of the safety factor (Km Animal) inthe considered animal and of the safety factor for humans (Km Human). KmHuman is equal to 37 and Km Rat is equal to 6.

The results are presented hereinafter.

Locomotion at the Center of the Test Space, Day PPD46; Effect of theSupplement on Anxiety

The results are represented in FIG. 8 .

In FIG. 8 : Effects of the treatment on anxiety. N=12; *** p<0.0001 withrespect to the treated group NS/vehicle; ####p<0.0001 with respect tothe treated group SP/vehicle; Dunnett test.

The group SP/Vehicle, corresponding to the individuals having beensubjected to a prenatal stress and preventively treated with the vehiclealone, has a percentage of displacements that is quite significantlyhigher within the peripheral area of the open test space in comparisonwith the group NS/Vehicle (the group that has not been subjected to aprenatal stress).

The group SP/supplement, corresponding to the individuals having beensubjected to a prenatal stress and preventively treated with thesupplement has a percentage of displacements that is quite significantlylower within the peripheral area of the open test space in comparisonwith the group SP/Vehicle (the group that has been subjected to aprenatal stress but not treated with the supplement). In addition, thepercentage of displacements of the group SP/supplement is equivalent tothat of the control group NS/vehicle.

A rate of displacements of the individuals in the peripheral area of theopen test space that is higher than the control modality is thedemonstration of an anxious behavior [63], via a protection mechanismbased on the search for boundaries limiting the uncovered areas and isto be monitored.

Thus, the prenatal stress (PS) has induced a very significant anxiousbehavior.

Surprisingly, it turns out that the supplement has quite significantlyand completely attenuated the anxious behavior induced by the prenatalstress.

Recognition Test, Day PPD47; Effect of the Supplement on the RecognitionMemory in the Recognition of an Object

The results are represented in FIG. 9 .

During this session, the same object is exhibited twice to theindividuals.

No statistical effect between the groups has been measured for thisparameter.

Thus, the individuals of all groups have interacted in an equivalentmanner when put in contact with identical objects and theirinteractions, both in terms of frequency and duration are equallydistributed between the two objects (50%).

Recognition Test, Day PPD48 (New Object); Effect of the Supplement onthe Recognition Memory for the Test of Recognition of a New Object

The results are represented in FIG. 10 .

In FIG. 10 : N=12; *** p<0.0001 with respect to the treated groupNS/vehicle; ###p<0.0001 with respect to the treated group SP/vehicle;Dunnett test.

During this session, two different objects are exhibited to theindividuals each once: one of the objects corresponds to the objectexhibited during the session 2 and the other object is a new object.

The group SP/vehicle, corresponding to the individuals having beensubjected to a prenatal stress and preventively treated with the vehiclealone, has a percentage of interactions, both in terms of frequency andduration, with the exhibited new object that is quite significantlylower in comparison with the group NS/Vehicle (the group that has notbeen subjected to a prenatal stress). And this percentage is equal tothat of the session 2 obtained for all groups. Thus, the individuals ofthe group SP/vehicle have as many interactions with the ancient objectas with the new object and therefore the individuals of this group donot recognize the ancient object exhibited during the session 2.

In contrast, the group SP/Supplement corresponding to the individualshaving been subjected to a prenatal stress and preventively treated withthe supplement, has a percentage of interactions, both in terms offrequency and duration, with the exhibited new object that is quitesignificantly higher in comparison with the group PS/Vehicle (thenegative control group). And this percentage is higher than that of thesession 2 obtained for all groups. Thus, the individuals of the groupSP/Supplement have less interactions with the ancient object than withthe new object, and therefore the individuals of this group recognizethe ancient object exhibited during the session 2. In addition, theindividuals of the group SP/Supplement have a percentage ofinteractions, both in terms of frequency and duration, with theexhibited new object that is equivalent with those of the groupsNS/Vehicle and NS/Supplement.

Thus, the prenatal stress (PS) has induced very considerable recognitionmemory deficits in the case of the new object.

Surprisingly, it turns out that the supplement has allowed attenuatingquite significantly and completely the recognition memory deficitsinduced by the prenatal stress.

In conclusion:

The treatment with the supplement has significantly and completelyattenuated the anxious behavior as well as the recognition memorydeficits induced by the prenatal stress.

The prenatal stress as practiced in this experiment significantlyinduces an anxious behavior, and quite significantly alters therecognition memory in young female rats.

Thus, by applying the formula for calculating the human equivalent dailydose, a preventive treatment attenuating the cognitive disorders causedby a prenatal stress may be defined with the daily intake of 0.05 to 0.1mg of supplement/kg of body weight.

Example 4: Test of a Natural Extract of the Microalga Phaeodactylumtricornutum in the Context of the In Vivo Model on the Attenuation ofthe Deficits Induced by the Age-Related Cognitive Decline

The food supplement of the disclosure is prepared from a Phaeodactylumtricornutum extract which comprises in mg/g:

-   -   Omega-3 type fatty acids (ALA, SDA, EPA, DHA): 66.6±11.5;    -   Fucoxanthin: 20.0±4.0;    -   Sterols: 3.0±0.6;    -   Phycoprostane: 0.0025±0.0005.

The supplement is obtained by addition of coconut oil in a proportion of410 mg±20 mg/g to said extract.

The supplement is incorporated into kibbles according to 4 differentformulations such that the incorporated amounts of the supplement withinthe different batches of kibbles correspond to human equivalent dailydoses as described in Table 2, and that, by dilution of the compositiondescribed hereinbelow in coconut oil with an equal final mass for allformulations.

The calculation of the human equivalent daily dose, from the daily dosetested in mice is defined as follows by the FDA (Guidance, 2005): thedaily dose in human expressed in mg/kg of body weight (HED Human) isequal to the daily dose in the animal expressed in mg/kg of body weight(HED Animal) multiplied by the ratio of the safety factor (Km Animal) inthe considered animal and of the safety factor for humans (Km Human). KmHuman is equal to 37 and Km Mice is equal to 3.

An additional batch of kibbles is formulated only with the coconut oil,such that the vehicle concentration is equivalent to that of the otherbatches, namely 0.01% (w:w).

The five batches of kibbles thus obtained are referenced as described inTable 2 hereinbelow.

TABLE 2 Human equivalent supplement dose (mg of supplement/ Formulatedkibbles kg of body mass/day) Reference Coconut oil (vehicle) 0 VehicleSupplement 1.7 D1 Supplement 3.3 D2 Supplement 4.2 D3 Supplement 5.3 D4

The considered in vivo model is the D-Galactose model applied to micewhich is suitable for the study of the age-related cognitive decline.Indeed, this model mimics numerous behavioral and molecularcharacteristics of the cerebral ageing in rodents' models.

The D-Galactose is administered subcutaneously in a daily proportion of150 mg/kg of mice wet weight, and the food supplement hereinabove isincorporated into a pellet, according to the following pattern:

-   -   Between day 28 and day 51, the supplement is administered by        incorporation into food pellets;    -   Between day 01 and day 51, the D-Galactose is administered        subcutaneously, five days a week;    -   Between days 43 and 51, three different behavioral tests are        used to monitor the effects of the test compounds.

The effectiveness of the supplement is assessed according to thefollowing parameters: improvement of the learning deficits (spatialworking memory: spontaneous alternation in the Y labyrinth according tothe Y-maze test; spatial memory by the so-called «Morris Water Maze» andlong-term contextual memory in the passive avoidance test), lipidperoxidation (LPO) rate in the hippocampus and effect on theneuro-inflammation markers IL6 and TNFα.

Improvement of the Learning Deficits

-   -   On day 43, all animals have been tested for the spontaneous        alternation performance in the Y-maze (YM) test, via a spatial        working memory index;    -   From day 44 to day 49, all animals have been tested for the        spatial memory in the Morris Water Maze (MWM) test, via a        spatial memory index;    -   From day 44 to day 49, all animals are tested via the MWM test        to assess the spatial working memory;    -   On days 50 and 51, the long-term contextual memory of the        animals is assessed using the step-by-step type passive        avoidance process (STPA), through exercise and retention        sessions, respectively.

Lipid Peroxidation (LPO) Rate in the Hippocampus and Effect on theNeuro-Inflammation Markers IL6 and TNFα

On the 51th day, after the behavioral tests, the animals have beeneuthanized.

For all animals, trunk blood is collected and centrifuged to recoverplasma and the brain is rapidly collected. The hippocampus and thecortex are dissected, the hippocampus is then used to determine thelipid peroxidation rates by a colorimetric method; the hemi-frontalcortex and the plasma are used to determine the level of theinflammatory biomarkers interleukin-6 (IL-6) and tumor necrosis factoralpha (TNF-α)

The quantification of the lipid peroxidation (LPO) rates has beencarried out according to the modified and adapted procedure ofHermes-Lima et al. This method measures the capacity of the peroxidizedlipids of the brain to oxidize a ferrous oxide and xylenol orangecomplex, set out in the presence of cumene hydroperoxide (HPC). Thelipid peroxidation level is determined in HPC-equivalent according tothe formula:

HPCE=A5801/A5802×[HPC (nmol)]

-   -   and expressed in HPC-equivalent per wet tissue weight and in        percentage with respect to the data obtained for the control        group (D-Galactose+vehicle).

The IL6 and TNFα contents are quantified by means of ELISA tests withthe following kits:

For the quantification of IL6: ThermoScientifique, EM2IL6

For the quantification of TNFα: ThermoScientifique, EMTNFA

For all tests, the cortex is homogenized after defrosting in a buffer of50 mM Tris-150 mM NaCl, pH 7.5, and sonicated for 20 s. Aftercentrifugation (16 100 g for min, 4° C.), a supernatant or plasma areused for the ELISA tests in compliance with the instructions of themanufacturer of the ELISA tests. For each test, the absorbance is readat 450 nm and the concentration of the sample is calculated using thestandard curve. The results are expressed in pg of marker per mg of wettissue.

All values, except the passive avoidance latencies, are expressed as anaverage more or less the standard deviation of the measurement.Statistical analyses are performed separately for each compound using aunidirectional ANOVA (value F), followed by a Dunnett post-hoc multiplecomparison test. The passive avoidance latencies do not follow aGaussian distribution, since the upper limit times are fixed. Hence,they are analyzed using a Kruskal-Wallis non-parametric ANOVA (value H),followed by Dunn multiple comparison test. The values with p<0.05 areconsidered as statistically significant.

The tests are performed on 72 male mice, distributed in 6 groups of 12mice, amongst which the group 1 is the negative control group and thegroups 2-6 are the positive control groups:

-   -   the group 1 is the group to which a subcutaneous saline solution        is administered instead of D-Galactose and kibbles B1;    -   the group 2 is the group to which D-Galactose and kibbles B1 are        administered;    -   the group 3 is the group to which D-Galactose and kibbles B2 are        administered;    -   the group 4 is the group to which D-Galactose and kibbles B3 are        administered; and    -   the group 5 is the group to which D-Galactose and kibbles B4 are        administered; and    -   the group 6 is the group to which D-Galactose and kibbles B5 are        administered.

Effects on the Spatial Memory in the Y-Maze Spontaneous AlternationTest:

The results are represented in FIG. 11 , the first diagram (to the left)illustrating the effects of the supplement of the disclosure on thespontaneous alternation deficits and the second diagram (to the right)illustrating the effects of the supplement of the invention on thelocomotor activity.

In FIG. 11 : Sol. Saline/veh corresponds to the negative control (thegroup that has not been treated with D-galactose and fed with thekibbles formulated with the vehicle, coconut oil); DGal 150/Vehcorresponds to the positive control (the group that has been treatedwith D-Galactose and fed with the kibbles formulated with the vehicle,coconut oil); D1, D2, D3 and D4 increasing doses of the supplement; N iscomprised between 11 and 12 depending on the groups; * p<0.05, ***p<0.0001 vs. the saline solution/group Veh, #p<0.05, ##p<0.01,###p<0.0001 vs. the group D-GAL 150/group Veh; Dunnett test.

It is observed that the treatment with D-Galactose has significantlyaltered the spatial working memory, in comparison with the mice treatedwith the saline solution.

The supplement D1 has quite significantly but partially attenuated thedeficits induced by the chronic intoxication with D-Galactose. Thesupplements D2, D3 and D44 have quite significantly and completelyattenuated the deficits induced by the chronic intoxication withD-Galactose.

Effects on the Learning Deficits Induced by the D-Gal According to theMWM Test:

The results are represented in FIG. 12 .

In FIG. 12 : Sol. Saline/veh corresponds to the negative control (thegroup that has not been treated with D-galactose and fed with thekibbles formulated with the vehicle, coconut oil); DGal 150/Vehcorresponds to the positive control (the group that has been treatedwith D-Galactose and fed with the kibbles formulated with the vehicle,coconut oil); D1, D2, D3 and D4 increasing doses of the supplement; N iscomprised between 11 and 12 depending on the groups; * p<0.05, **p<0.01, *** p<0.0001 vs. the saline solution/group Veh, ##p<0.01,###p<0.0001 vs. the group D-GAL 150/group Veh; Bonferroni multiplecomparison test after bidirectional ANOVA.

The chronic intoxication with D-Galactose has considerably altered thespatial learning, in comparison with the negative control group (salinesolution/vehicle).

The supplement D1 has quite significantly but partially attenuated thedeficits induced by the chronic intoxication with D-Galactose.

The supplements D2, D3 and D4 have quite significantly and completelyattenuated the deficits induced by the chronic intoxication withD-Galactose.

Effects of the Supplement on the Learning Deficits Induced by theD-Galactose

The results are represented in FIG. 13 .

In FIG. 13 : Sol. Saline/veh corresponds to the negative control (thegroup that has not been treated with D-galactose and fed with thekibbles formulated with the vehicle, coconut oil); DGal 150/Vehcorresponds to the positive control (the group that has been treatedwith D-Galactose and fed with the kibbles formulated with the vehicle,coconut oil); D1, D2, D3 and D4 increasing doses of the supplement; N iscomprised between 11 and 12 depending on the groups; Sol. Saline/vehcorresponds to the negative control (the group that has not been treatedwith D-galactose and fed with the kibbles formulated with the vehicle,coconut oil); DGal 150/Veh corresponds to the positive control (thegroup that has been treated with D-Galactose and fed with the kibblesformulated with the vehicle, coconut oil); D1, D2, D3 and D4 increasingdoses of the supplement; N is comprised between 11 and 12 depending onthe groups; *** p<0.0001 vs. the saline solution/group Veh, /Veh;###p<0.0001 vs. the group D-GAL 150/Veh; Bonferroni multiple comparisontest after bidirectional ANOVA. “T”, the time spent in the targetquadrant; “0”, the average time spent in the three other quadrants.

The chronic intoxication with D-Galactose has considerably altered thespatial learning, in comparison with the negative control group (salinesolution/vehicle).

The supplements D1 and D2 have quite significantly but partiallyattenuated the deficits induced by the chronic intoxication withD-Galactose.

The supplements D3 and D4 have quite significantly and completelyalleviated the deficits induced by the chronic intoxication withD-Galactose.

Effects on the Passive Avoidance Deficits Induced by the D-Galactose inMice

The results are represented in FIG. 14 , with the effects of thesupplement of the disclosure on the step-down latency illustrated on theleft-side diagram and on the escape latency illustrated on theright-side diagram, measured during the retention period.

In FIG. 14 : Sol. Saline/veh corresponds to the negative control (thegroup that has not been treated with D-galactose and fed with thekibbles formulated with the vehicle, coconut oil); DGal 150/Vehcorresponds to the positive control (the group that has been treatedwith D-Galactose and fed with the kibbles formulated with the vehicle,coconut oil); D1, D2, D3 and D4 increasing doses of the supplement; N iscomprised between 11 and 12 depending on the groups; *** p<0.0001 vs.the saline solution/group Veh, ###p<0.0001 vs. the group D-GAL 150/groupVeh; Dunnett test.

The chronic intoxication with D-Galactose has considerably altered thelong-term contextual working memory, in comparison with the negativecontrol group (saline solution/vehicle).

The supplement D1 has not demonstrated any effect on the long-termcontextual memory.

The supplements D2, D3 and D4 have quite significantly and completelyattenuated the deficits induced by the chronic intoxication withD-Galactose.

Effects of the Supplement on the Lipid Peroxidation Induced by theD-Galactose

The results are represented in FIG. 15 .

In FIG. 15 : Sol. Saline/veh corresponds to the negative control (thegroup that has not been treated with D-galactose and fed with thekibbles formulated with the vehicle, coconut oil); DGal 150/Vehcorresponds to the positive control (the group that has been treatedwith D-Galactose and fed with the kibbles formulated with the vehicle,coconut oil); D1, D2, D3 and D4 increasing doses of the supplement; N iscomprised between 11 and 12 depending on the groups; ** p<0.01, ***p<0.0001 vs. the saline solution/group Veh, ##p<0.01, ###p<0.0001 vs.the group D-GAL 150/group Veh; Dunnett test.

The chronic intoxication with D-Galactose has considerably increased theoxidative stress, in comparison with the negative control group (salinesolution/vehicle).

The supplement D1 has quite significantly but partially reduced theoxidative stress induced by the chronic intoxication with D-Galactose.

The supplements D2, D3 and D4 have quite significantly and completelyreduced the oxidative stress induced by the chronic intoxication withD-Galactose.

Effects of the Supplement on the Induced Expression of TNF-α in theCortex and the Plasma by the D-Galactose

The results are represented in FIG. 16 , with the effect on the cortexon the left-side diagram and the effect on the plasma on the right-sidediagram.

In FIG. 16 : Sol. Saline/veh corresponds to the negative control (thegroup that has not been treated with D-galactose and fed with thekibbles formulated with the vehicle, coconut oil); DGal 150/Vehcorresponds to the positive control (the group that has been treatedwith D-Galactose and fed with the kibbles formulated with the vehicle,coconut oil); D1, D2, D3 and D4 increasing doses of the supplement; N iscomprised between 11 and 12 depending on the groups; *** p<0.0001 vs.the saline solution/group Veh, ###p<0.0001 vs. the group D-GAL 150/groupVeh; Dunnett test.

The chronic intoxication with D-Galactose has considerably increased ina significant manner the TNF-α in the cortex and the plasma, incomparison with the negative control group (saline solution/vehicle).

The supplement D1 has quite significantly but partially reduced theincrease of the TNF-α induced by the chronic intoxication with D-Gal inthe brain and the plasma.

The supplement D2 has quite significantly and completely reduced theincrease of the TNF-α induced by the chronic intoxication with D-Gal inthe brain, but partially in the plasmas.

The supplement D3 has quite significantly but partially reduced theincrease of the TNF-α induced by the chronic intoxication with D-Gal inthe brain, and completely in the plasma.

The supplement D4 has quite significantly and completely reduced theincrease of the TNF-α induced by the chronic intoxication with D-Gal inthe brain and the plasma.

Effects of the Supplement on the Induced Expression of IL-6 in theCortex and the Plasma by the D-Galactose

The results are represented in FIG. 17 , with the effect on the cortexon the left-side diagram and the effect on the plasma on the right-sidediagram.

In FIG. 17 : Sol. Saline/veh corresponds to the negative control (thegroup that has not been treated with D-galactose and fed with thekibbles formulated with the vehicle, coconut oil); DGal 150/Vehcorresponds to the positive control (the group that has been treatedwith D-Galactose and fed with the kibbles formulated with the vehicle,coconut oil); D1, D2, D3 and D4 increasing doses of the supplement; N iscomprised between 11 and 12 depending on the groups; *** p<0.0001 vs.the saline solution/group Veh, ###p<0.0001 vs. the group D-GAL 150/groupVeh; Dunnett test.

The chronic intoxication with D-Galactose has considerably increased ina significant manner the IL-6 in the cortex and the plasma, incomparison with the negative control group (saline solution/vehicle).

The supplements D1 and D2 have quite significantly but partially reducedthe increase of the IL-6 induced by the chronic intoxication with D-Galin the brain and the plasmas.

The supplements D3 and D4 have quite significantly and completelyreduced the increase of the IL-6 induced by the chronic intoxicationwith D-Gal in the brain and the plasma.

IN CONCLUSION

The chronic intoxication with D-Galactose has quite significantlyinduced an alteration of the spatial working memory, of the long-termcontextual memory and has prejudiced the spatial learning. Thebehavioral alterations are also related to biochemical alterationsmanifested in an increase of oxidative stress and in an induction ofneuro-inflammatory processes.

The preventive treatment with the supplement of the invention isdose-dependent and has quite significantly and completely attenuated inthe case of the strongest tested dose (the supplement D4) the deficitsinduced by the chronic intoxication with D-Galactose manifested in abehavioral alteration, an increase of oxidative stress and an activationof neuro-inflammatory processes. And in the cases of the intermediatelower doses (the supplements D2 and D3), the supplement of the inventionhas quite significantly and completely attenuated the deficits inducedby the chronic intoxication with D-Galactose manifested in the spatialworking memory, an increase of oxidative stress and an activation ofneuro-inflammatory processes.

Thus, by applying the formula for calculating the human equivalent dailydose, a preventive treatment of the age-related cognitive decline may bedefined with the daily intake of 1.7 to 5.3 mg of supplement/kg of bodyweight.

Example 5: Tests of a Natural Extract of the Microalga Phaeodactylumtricornutum in a Human Clinical Trials on the Attenuation of theDeficits Induced by the Age-Related Cognitive Decline

The food supplement of the disclosure is prepared from the waterinsoluble Phaeodactylum tricornutum extract obtained by solid-liquidextraction as described in Example 1 and comprises in mg/g:

-   -   Omega-3 type fatty acids (ALA, SDA, EPA, DHA): 33.3±5;    -   Fucoxanthin: 10.0±2;    -   Sterols: 1.5±0.3;    -   Phycoprostane: 0.00125±0.00025.

Two double blind, randomized manner, human clinical trials have beenconducted as detailed below.

Trial 1:

A population of 40 free living males and females aged between 55 to 75years with age-associated memory decline have been administrated orally4 capsules containing 275 mg of the food supplement above or 4 capsulesof a placebo (275 mg of maltodextrine) daily during a period of 12weeks. Cognitive function battery has been realized after 4 and 12 weeksof daily supplementation. The inclusion and exclusion criteria were asbelow:

Inclusion Criteria:

Be able to give written informed consent and to consume theinvestigational product daily for the duration of the study.

Healthy males and females aged ≥55 and ≤75 years old.

-   -   Body-mass index between 18.5 and 35.0 kg/m 2    -   Is free-living (living in a private home, alone or with family,        and able to maintain their health and hygiene without        assistance).    -   Have age-associated memory impairment (AAMI) based on the        following National Institute of Mental Health criteria,        specified as scoring:        -   1. Absence of dementia as determined by a score of >24 on            the Mini Mental State Examination (MMSE).        -   2. ≤29 or ≤9 on the VPA I and II portions of the Wechsler            Memory Scale IV, respectively        -   3. A score on the MAC-Q of 25.    -   Is in general good health, as determined by the investigator    -   Ability to comply and understand the cognitive function practice        tests    -   Willing to maintain their habitual diet and exercise routines.    -   Willing to maintain consistent sleep duration the evening before        study visits.

Exclusion Criteria:

-   -   Use of medications or supplements known to alter cognitive        function within past 2 weeks    -   Abnormal clinical laboratory test that may affect study outcome.    -   History or presence of clinically important cardiac, renal,        hepatic, endocrine, pulmonary, biliary, gastrointestinal,        pancreatic, or neurological disorders (including sleep        disorders, head injuries, Alzheimer's disease, Parkinson's        disease, stroke, inflammatory brain disease    -   History or presence of cancer, except nonmelanoma skin cancer    -   Uncontrolled hypertension/diabetes    -   History of depression within past 24 months or use of        psychotropic medications within 1 month of screening    -   Planned major changes in lifestyle (i.e. diet, dieting, exercise        level, travelling) during the duration of the study.    -   History within previous 12 months of alcohol or substance abuse.    -   History of heavy smoking (>1 pack/day) within past 3 months.

The specific cognitive functions evaluated after 4 and 12 weeks of dailysupplementation were as follows:

-   -   Change in spatial Working Memory, using a Mean Span Score in        Corsi Blocks module of the COMPASS (Computerized Pilot Aptitude        Screening System) (Kesseis, R. P. C.; van Zandvoort, M. J. E.;        Postma, A.; Kappelle, L. J.; de Haan, E. H. F (2000). “The Corsi        Block-Tapping Task: Standardization and Normative Date. Applied        Neuropsychology, 7 (4): 252-258)    -   Change in attention and vigilance, evaluated by Mean reaction        time and accuracy through Digit Vigilance Task module of the        COMPASS    -   Change in executive function, evaluated by Mean reaction time        and accuracy through Stroop task module of the COMPASS (Stoop,        John Ridley (1935) “Studies of interference in serial verbal        reactions”, Journal of Experimental Psychology, 18 (6): 643-662,        doi:10.1037/h0054651)    -   Change in episodic memory, evaluated by Mean reaction time and        accuracy through Stroop task module of the COMPASS (Stoop, John        Ridley (1935) “Studies of interference in serial verbal        reactions”, Journal of Experimental Psychology, 18 (6): 643-662,        doi:10.1037/h0054651)    -   Change in perceptual and cognitive skills, evaluated by Mean        reaction time and accuracy through Neurotracker light reaction        test    -   Change in Interleukin-6 level (pg/ml) and hs-CRP (Immunodosage        ELISA).

C-Reactive Protein (CRP) is an acute-phase protein, produced exclusivelyin the liver. Interleukin-6 is the mediator for the synthesis by thehepatocytes of CRP. It's well admitted in scientific literature thatelevated levels of blood CRP predict poorer cognitive function andincreased dementia risk in cognitively healthy middle-aged and olderadults. (Lewis, N. A., and Knight, J. E. (2021)). Longitudinalassociations between C-reactive protein and cognitive performance innormative cognitive ageing and dementia. Age Ageing 50, 2199-2205. doi:10.1093/ageing/afab152).

A volume of 21.5 mL of blood of the individuals of the 2 groups werecollected, frozen and further, the following have been measured:

-   -   Safety Profile    -   IL-6    -   TNF-α    -   IFN-α    -   C-reactive protein (CRP)    -   Insulin    -   HbA1C (Glycated hemoglobin)    -   Computerised Corsi Blocks Task

All computerised cognitive/mood assessments were identical, and werecarried out via laptop computers and response boxes using theComputerised Mental Performance Assessment System (COMPASS, NorthumbriaUniversity, Newcastle upon Tyne, UK). This software platformincorporates the presentation of classic and custom computerisedcognitive tasks, with fully randomised parallel versions of each taskdelivered at each assessment for each individual. A similar selection oftasks has previously been shown to be sensitive to diverse nutritionalinterventions.

-   Stonehouse W., Conlon C. A., Podd J., Hill S. R., Minihane A. M.,    Haskell C., Kennedy D. DHA supplementation improved both memory and    reaction time in healthy young adults: A randomized controlled    trial. Am. J. Clin. Nutr. 2013; 97:1134-1143. doi:    10.3945/ajcn.112.053371. [PubMed] [CrossRef] [Google Scholar]-   Kennedy D. O., Jackson P. A., Forster J., Khan J., Grothe T.,    Perrinjaquet-Moccetti T., Haskell-Ramsay C. F. Acute effects of a    wild green-oat (Avena sativa) extract on cognitive function in    middle-aged adults: A double-blind, placebo-controlled,    within-subjects trial. Nutr. Neurosci. 2017; 20:135-151. doi:    10.1080/1028415X.2015.1101304. [PubMed] [CrossRef] [Google Scholar]-   Kennedy D., Wightman E., Khan J., Grothe T., Jackson P. The Acute    and Chronic Cognitive and Cerebral Blood-Flow Effects of Nepalese    Pepper (Zanthoxylum armatum DC.) Extract—A Randomized, Double-Blind,    Placebo-Controlled Study in Healthy Humans. Nutrients. 2019;    11:3022. doi: [PMC free article] [PubMed] [CrossRef] [Google    Scholar]-   Kennedy D. O., Wightman E. L., Forster J., Khan J.,    Haskell-Ramsay C. F., Jackson P. A. Cognitive and mood effects of a    nutrient enriched breakfast bar in healthy adults: A randomised,    double-blind, placebo-controlled, parallel groups study. Nutrients.    2017; 9:1332. doi: 10.3390/nu9121332. [PMC free article] [PubMed]    [CrossRef] [Google Scholar]-   Kennedy D., Okello E., Chazot P., Howes M.-J., Ohiomokhare S.,    Jackson P., Haskell-Ramsay C., Khan J., Forster J., Wightman E.    Volatile terpenes and brain function: Investigation of the cognitive    and mood effects of Menthax piperita I. essential oil with in vitro    properties relevant to central nervous system function. Nutrients.    2018; 10:1029. doi: 10.3390/nu10081029. [PMC free article] [PubMed]    [CrossRef] [Google Scholar]

Nine blue squares on a black background are displayed on the screen.Some of the blue squares change to red and back to blue again in asequence. Participants were required to remember this sequence. The taskwas repeated five times at each level of difficulty with the sequencespan increasing from 4 upwards, until the participant could no longercorrectly recall the sequences. For Corsi blocks and Corsi blocks lite,participants used the cursor to click the blocks in the exact sequencein which they were presented. For reversed Corsi blocks, they clicked onthe blocks in reverse to the sequence shown.

Corsi blocks and reversed Corsi blocks were continued up to fifteenlevels (i.e. up to squares in each sequence), as long as participantswere making enough correct responses. As soon as they made less than 3correct responses (out of the five in one level) the task ended. Corsiblocks lite is a shorter task with a maximum of 6 blocks in a sequence;all participants completed levels 4-6 regardless of incorrect responses.This module is particularly useful for training configurations.

The tests are described below:

Choice Reaction Time Task

Arrows pointing left and right appear on the screen at irregularintervals. The participant is required to indicate the direction of thearrow as quickly as possible, whenever an arrow is displayed.

Digit Vigilance Task

A fixed number appear on the right of the screen and a series ofchanging numbers appear on the left side of the screen. Participants arerequired to make a response when the number on the left matches thenumber on the right.

Stroop Task

A series of colour names (RED, YELLOW, GREEN, BLUE) are displayed on thescreen. These are written in a coloured font. Participants are requiredto make a response based on the colour font the word is written in notthe colour the word depicts. Participants are shown either congruent orincongruent stimuli, or a random mix of both.

Picture Presentation Task

A series of pictures are displayed on the screen, one at a time. Thenumber of pictures, the rate at which they are displayed and theinterstimulus interval can be modified.

Picture Recognition Task

All target pictures shown during Picture Presentation plus an equalnumber of decoys will be displayed on the screen one at a time. For eachstimulus, participants select ‘Yes’ or ‘No’ to indicate if they haveseen the picture before or not.

Word Recognition Task

All target words that were shown during Word Presentation plus an equalnumber of decoys will be displayed on the screen one at a time.Participants indicate if they remember seeing the word earlier or not.

Word Presentation/Word Recall (Immediate and Delayed) Task

Word Presentation

A list of words is displayed on the screen, one word at a time. Thenumber of words, the rate at which they are displayed and theinterstimulus interval can be modified.

Word recall (scored manually, see Processing Word Recall Data documentfor details)

Participants are given 60 seconds to write down as many of the wordsthey were shown earlier as they can. This task can be performedimmediately following presentation of the words (Immediate recall) orfollowing a delay (Delayed recall).

Bond-Lader Mood Rating Scale

This is a series of mood scales. Each scale is a line anchored at eitherside by an adjective describing a mood. Participants must click (usingthe mouse) at a point on the scale that represents how they are feelingat that point in time. There are 16 scales in total, each scored out of100. From these scales three composite scores are calculated describingfeelings of ‘Alert’, ‘Calm’ and ‘Content’, which are also presented as ascore out of 100.

Trial 2: A population of 66 healthy individuals male and female, agedbetween 55 and 75 years old, with age-associated memory decline havebeen administrated orally 2 capsules containing 275 mg of the foodsupplement above or 2 capsules of a placebo (275 mg of maltodextrine)daily during a total period of 24 weeks. Cognitive function batterytests have been realized after 12 and 24 weeks of daily supplementation.The inclusion and exclusion criteria were the same as for the trialdescribed above.

The blood concentration of CRP was measured in the population after 24weeks of daily supplementation with the food supplement, as previouslydescribed in trial 1 (ELISA technique).

The results demonstrated a significant reduction of inflammation byreduction of the concentration of hs-CRP after 24 weeks in the group whoreceived the food supplement according to the invention versus theplacebo group.

1. A food supplement comprising a water-insoluble extract of a microalgaand at least one oil selected from medium-chain triglycerides (MCT),wherein the extract of microalga comprises at least 25 mg/g of one orseveral omega-3 type fatty acid(s), at least 5 mg/g of one or severalxanthophyll(s), at least 0.5 mg/g of one or several sterol(s) and atleast 1 μg/g of one or several phycoprostane(s).
 2. The food supplementaccording to claim 1, wherein the extract comprises 25 to 250 mg/g ofone or several omega-3 type fatty acid(s), 5 to 50 mg/g of one orseveral xanthophyll(s), 0.5 to 20 mg/g of one or several sterol(s), 1 to100 μg/g of one or several phycoprostane(s).
 3. The food supplementaccording to claim 1, wherein the extract comprises 25 to 200 mg/g ofone or several omega-3 type fatty acid(s), 5 to 30 mg/g of one orseveral xanthophyll(s), 0.5 to 8 mg/g of one or several sterol(s) and 1to 50 μg/g of one or several phycoprostane(s).
 4. The food supplementaccording to claim 1, wherein the extract comprises 25 to 170 mg/g ofone or several omega-3 type fatty acid(s), 5 to 25 mg/g of one orseveral xanthophyll(s), 0.5 to 6 mg/g of one or several sterol(s) and 1to 40 μg/g of one or several phycoprostane(s).
 5. The food supplementaccording to claim 1, wherein the medium-chain triglycerides (MCT) areselected from coconut palm oil and palm oil.
 6. The food supplementaccording to claim 1, wherein the or at least one of the omega-3 typefatty acids is selected from ALA (α-linolenic acid), stearidonic acid(SDA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), andmixtures thereof.
 7. The food supplement according to claim 1, whereinthe at least one of the xanthophylls is fucoxanthin.
 8. The foodsupplement according to claim 1, wherein the or at least one of thesterols is selected from phytosterols.
 9. The food supplement accordingto claim 1, wherein the or at least one of the phycoprostanes isselected from phytoprostanes, isoprostanes and neuroprostanes.
 10. Thefood supplement according to claim 1, which further comprises at leastone additive selected from preservative agents, colorants, flavors,disintegration agents, lubricant agents, coating or encapsulationagents.
 11. The food supplement according to claim 1, which is in theform of gel capsules, capsules, tablets, pastilles or loose powder. 12.The food supplement according to claim 1, which is packaged in doseshaving a unit weight comprised between 10 mg and 1 g.
 13. The foodsupplement according to claim 1, wherein the microalga is selected fromany one of the taxons Pinguiophyceae, Chrysophyceae, Bacillariophyceae,Mamiellophyceae, Prymnesiophyceae, Haptophyceae, Coccolithophyceae,lsochrysidaceae and Phaeodactylaceae.
 14. The food supplement accordingto claim 13, wherein said microalga is Tisochrysis lutea orPhaeodactylum tricornutum.
 15. A method for preventing the apparition ofage-related cognitive disorders, defined as a non-pathological decreaseof the cognitive functions, or cognitive disorders in children or youngadults having been subjected to a prenatal stress inducingnon-pathological disorders, such as hyperactivity, attention and memorydeficit, language retardation and anxious behavior, comprising theadministration of an effective amount of a food supplement according toclaim 1 to a human in need thereof.
 16. The method for preventing theapparition of age-related cognitive disorders, defined as anon-pathological decrease of the cognitive functions according to claim15, wherein the age-related cognitive disorders are chosen from deficitin spatial working memory, deficit in attention and vigilance, deficitin executive function, deficit in episodic memory, and deficit inperceptual and cognitive skills.
 17. The method for preventing theapparition of age-related cognitive disorders, defined as anon-pathological decrease of the cognitive functions according to claim16, in order to improve calmness and reduce perceived stress.
 18. Themethod for preventing the apparition of age-related cognitive disorders,defined as a non-pathological decrease of the cognitive functionsaccording to claim 15 comprising the administration of an effectiveamount of a food supplement according to claim 1 to a healthy olderhuman aged between 55 and 75 years old.
 19. The method for preventingthe apparition of age-related cognitive disorders, defined as anon-pathological decrease of the cognitive functions according to claim15, wherein the effective amount corresponds to a daily intake ofbetween 2 to 16 mg of food supplement/kg of body weight.
 20. The methodfor preventing the apparition of age-related cognitive disorders,defined as a non-pathological decrease of the cognitive functionsaccording to claim 19, wherein the food supplement is orallyadministrated during between 1 month to 6 months.
 21. The method forpreventing the apparition of cognitive disorders in children or youngadults having been subjected to a prenatal stress inducingnon-pathological disorders, such as hyperactivity, attention and memorydeficit, language retardation and anxious behavior according to claim15, wherein the effective amount corresponds to a daily intake ofbetween 0.05 to 0.1 mg of food supplement/kg of body weight.